​Stressed about Picking an Oxidative Damage Assay?​

2017-07-13

Find the right biomarker to detect in your application

Oxidative stress can be evaluated directly by measuring reactive oxygen species (ROS), or indirectly by the associated damage to lipids, proteins, and nucleic acids that occurs upon overproduction of ROS. Oxidation of these macromolecules is a contributing factor toward the onset of aging, cancer, cardiovascular, and inflammatory diseases. Although direct measurement of ROS is ideal, the indirect methods are often relied on more heavily due to the relative stability of damage markers on biomolecules compared to the transient nature of ROS. As such, peer reviewers often request confirmation of oxidative stress by testing multiple oxidative stress markers either by assessing multiple markers on the same biomolecule (e.g., different markers of lipid peroxidation) or by examining one marker each on proteins, lipids, and nucleic acids. Assay selection is also often driven by sample type since some oxidative stress markers are more easily detected in certain biofluids, whereas others are more readily detected in cells and tissues. To help you determine which is best to use in your experimental system, here is a breakdown of the assay technology used to detect the most common oxidative damage biomarkers.​​

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ROS (and RNS)

The four electron reduction of oxygen to water occurs normally as part of metabolism. During this process, the formation of various ROS, including hydrogen peroxide (H2O2) and superoxide (O2•−), can occur to be used as cell signaling molecules, or as the result of metabolic dysfunction. Damage to cellular macromolecules occurs ​when uncontrolled oxidation stresses a biological system. Assays for ROS do not discern the source of ROS production (i.e., normal vs. disease state), but if the experimental model is under stress, an increase in ROS and alteration to molecular components is probable.

H2O2 is a relatively stable ROS that can be detected using fluorescent probes such as ADHP (10-acetyl-3,7-dihydroxy​​​phenoxazine) when coupled to a peroxidase. Assay specificity is improved significantly when an H2O2 scavenger, such as catalase, is included as a control. Xanthine oxidase (XO), the terminal enzyme of purine catabolism (hypoxanthine → xanthine → uric acid) in humans, is a critical source of ROS in several disease states. It produces both H2O2 and O2•− at a ratio of 4:1 at 21% O2 and pH 7.0.1 The activity of this enzyme can be measured by allowing XO to degrade hypoxanthine and capturing the H2O2 byproduct of this reaction via a probe like ADHP coupled to a peroxidase, such as HRP. Dihydroethidium (hydroethidine or DHE) is another widely used redox-sensitive probe that can be used directly in live cells. This probe is oxidized by O2•− to form 2-hydroxyethidium (ex 500-530 nm/em 590-620 nm) or by non-specific oxidation by H2O2 or other sources of ROS to form ethidium (ex 480 nm/em 576 nm).2,3 Note that the spectral range between 2-hydroxyethidium and ethidium is narrow, so O2•− can be distinguished from H2O2 with appropriate monochromator-based optical systems.

Reactive nitrogen species (RNS) are also produced during oxidative stress. High levels of O2•− and nitric oxide (NO), synthesized by nitric oxide synthase (NOS), can lead to the formation of peroxynitrite. NO itself also reacts with thiols and iron-sulfur enzymes, whereas peroxynitrite reacts with tyrosine residues to form nitrotyrosine. Nitrite (NO2) and nitrate (NO3) are end products of in vivo NO reactions whose total production can be detected using either Griess reagents or DAN.4,5 Because the relative proportion of NO2 and NO3 is variable, these assays first convert NO3 to NO2 using NADPH-dependent nitrate reductase. Subsequent reaction with Griess reagents or DAN, both of which only react with NO2, will determine a total concentration of NO2. Detection of NOS activity in tissues and cells can be accomplished by harnessing the NOS-driven conversion of a radiolabeled arginine to citrulline in the presence of the necessary factors (i.e., oxygen, FMN, FAD, NADPH+, calcium, calmodulin, and tetrahydrobiopterin). Alternatively, in vitro NOS activity can be detected using the chemistry of the Griess reaction once the excess NADPH added as a cofactor for NOS activity is removed using an oxidization step that is catalyzed by lactate dehydrogenase.

DNA/RNA Damage

Guanine is the base that is most prone to oxidation when DNA and RNA are damaged. The repair processes that are initiated to correct this damage release multiple oxidized guanine species into the urine. These species include the ribose-free base (8-hydroxyguanine), the nucleoside from RNA (8-hydroxyguanosine), and the deoxynucleoside from DNA (8-hydroxy-2’-deoxyguanosine). Historically, 8-hydroxy-2'-deoxyguanosine was the most accepted biomarker for oxidative damage to DNA, yet in recent years it has become increasingly understood that RNA damage markers should also be assessed. 8-Hydroxyguanosine is reported to be a better marker of age-related oxidant damage,6,7 and 8-hydroxyguanine is a better marker in certain cancer patients.8 Thus, assays that can detect multiple oxidized guanine species capture a more complete set of biologically relevant products of oxidative damage than do assays that are restricted to analysis of only one (e.g., 8-hydroxy-2'-deoxyguanosine).9

Protein Oxidation (and Nitration)

The most common marker of protein oxidation is protein carbonyl content.10 The presence of redox cycling cations like Fe2+ or Cu2+, in conjunction with attack by ROS, leads to the formation of amino acid derivatives containing carbonyl groups (ketones, aldehydes). Cigarette smoke and aldehydes also introduce carbonyls into proteins. A convenient technique to detect carbonyl content in protein preparations involves a reaction between 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls, which forms a Schiff base that produces a corresponding hydrazone that can be measured spectrophotometrically. Alternatively, ROS exposure to a protein’s methionine residues generates protein methionine sulfoxide (MetO), a reversible oxidative modification, that if not overturned by MetO reductases, is irreversibly oxidized to methionine sulfone. An antibody specific for protein methionine sulfoxide can be used to monitor this process by detecting proteins containing MetO residues.11

The presence of nitrotyrosine on proteins is used as a marker of peroxynitrite formed in vivo when NO reacts with O2•−.12 As peroxynitrite undergoes heterolytic cleavage, freed nitronium ions nitrate protein tyrosine residues. An antibody specific for nitrotyrosine can be used to detect protein nitration. NO can also directly modify proteins through the RNS-mediated process of S-nitrosylation wherein an NO group reacts with thiol groups of protein cysteine residues, resulting in the formation of an S-NO moiety. This reversible modification has been shown to regulate the function of various proteins. The most thoroughly validated method to directly visualize protein S-nitrosylation is termed the biotin switch technique.13 This method cleaves S-NO bonds (after blocking existing free thiols) to biotinylate the resulting newly formed free thiol groups. This technique can also be used to detect the oxidation of glutathione peptides produced by the process of S-glutathionylation (PSSG).

Lipid Peroxidation

Lipid peroxidation results in the formation of highly reactive and unstable hydroperoxides of unsaturated lipids. These hydroperoxides can be efficiently extracted and measured directly by utilizing redox reactions with ferrous ions to reveal the total hydroperoxide content present at a moment in time. However, the more traditionally used biomarkers of lipid peroxidation are the decomposition products (e.g., alkanes, ketones, aldehydes) of unstable hydroperoxides. Malondialdehyde (MDA) and 4-hydroxy nonenal (4-HNE) are the most well known degradants of polyunsaturated fatty acid (PUFA) hydroperoxides.14 MDA assays using a thiobarbituric reaction are named Thiobarbituric Acid Reactive Substances assays or TBARS assays, since thiobarbituric acid reacts various aldehydes produced during lipid peroxidation in addition to MDA. While the specificity of TBARS toward compounds other than MDA generates uncertainty, it remains the most widely used assay used to determine lipid peroxidation.15 4-HNE protein adducts are typically more stable than MDA protein adducts, so assays detecting this marker are sometimes preferred over TBARS depending on sample quality. 1,4-Dihydroxynonane mercapturic acid (DHN-MA), the major urinary metabolite of 4-HNE, is an additional biomarker that may be assayed. It should be noted that measurement of 4-HNE as an indirect indicator of lipid peroxidation will not provide a complete analysis since only hydroperoxides derived from ω-6 PUFAs (and not ω-3 PUFAs) give rise to this byproduct, and 4-HNE formation is variable depending on transition metal ion content. These factors, in addition to the specificity issue, can lead to an inaccurate estimation of lipid peroxidation.

8-Isoprostane, produced by random oxidation of tissue phospholipids, is currently considered one of the most reliable biomarkers of in vivo lipid peroxidation.16 It is a specific product of lipid peroxidation that is stable and levels are present in detectable quantities in all normal biological fluids and tissues. 8-Isoprostane is present both free and esterified in membrane phospholipids, so measurement of 8-isoprostane in urine will assess levels of the free form. Plasma or serum must be hydrolyzed to obtain total 8-isoprostane (membrane-bound and free) concentration. Measurement of levels esterified in phospholipids can be used to determine the extent of lipid peroxidation in target sites of interest. 8-Isoprostane is typically assessed using either an immunoassay or LC-MS or GC-MS, depending on instrumentation accessibility. Note that it is common for immunoassays to report higher analyte concentrations compared to LC-MS or GC-MS readouts. LC-MS or GC-MS analyses typically measure only a single compound, whereas antibodies used in immunoassays can potentially recognize the target molecule, biologically relevant metabolites, and other closely related isoprostane species. In some cases, measurement of a combination of these molecules offers a more complete representation of the overall biological response compared to the measurement of a short-lived parent molecule.

Available from Cayman

Cayman offers a broad portfolio of sensitive, easy-to-use assays and reagents to quantify ROS or the damage produced by ROS as evidenced by oxidation of DNA/RNA, proteins, and/or lipids. This also includes a collection of assays to assess distinct antioxidant mechanisms within the cell (e.g., ascorbic acid, catalase, glutathione, superoxide dismutase, and thioredoxins) that counteract the effects of ROS in vivo. Mass spectrometry standards, LC-MS mixtures, sample purification kits, validated antibodies, and selective probes are also available to assist in your determination of oxidative stress.​

RO​S (and RNS)

Item No.Product Name​Sample Type​MeasureMethodology​Additional Info
600050 Hydrogen Peroxide Cell-Based Assay KitCultured cellsExtracellular H2O2Plate-based fluorometric measurement (ex 530-560 nm, em 590 nm)Utilizes ADHP, a sensitive and stable probe for H2O2 and includes catalase to check assay specificity
780001 Nitrate/Nitrite Colorimetric Assay KitPlasma, serum, urine, tissue culture media, and tissue hom​ogenatesNO metabolitesPlate-based colorimetric measurement (540-550 nm)Uses a small amount of added NADPH in conjunction with a catalytic system for recycling spent NADP+ back to NADPH to avoid NADPH interference with the chemistry of the Griess reagents; works well for the analysis of fluids such as plasma and urine, but cannot be used to analyze NO2- and NO3- from an in vitro assay of NOS in which excess NADPH has been added
760871 Nitrate/Nitrite Colorimetric Assay Kit (LDH method)Plasma, serum, urine, and tissue homogenates In vitro NOS activity and NO metabolitesPlate-based colorimetric measurement (530-550 nm)Use to analyze NO2- and NO3- from an in vitro NOS assay in which excess NADPH has been added; an extra step is included in the protocol that uses LDH to remove the excess NADPH
780051 Nitrate/Nitrite Fluorometric Assay KitPlasma, serum, tissue culture media, and tissue homogenatesNO metabolitesPlate-based fluorometric measurement (ex 360-365 nm, em 430 nm)Utilizes DAN instead of Griess reagents, which enables 20-fold increased sensitivity over the colorimetric version; allows for detection of low concentrations of NO2- and NO3- (minimum detectable quantity of NO2/NO3 is ~50 nM)
781001 NOS Activity Assay KitCell lysates and purified preparationsNOS activityLiquid scintillation countingMonitors the conversion of radiolabeled arginine to citrulline by NOS
601290 ROS Detection Cell-Based Assay Kit (DHE)Live cellsROSFlow cytometer or plate-based fluorometric measurement (ex 480-520 nm, em 570-600 nm)Utilizes the redox-sensitive probe DHE as a substrate for O2•− and H2O2; includes positive control for ROS generation and negative control for ROS scavenging
10010895 Xanthine Oxidase Fluorometric Assay KitPlasma, serum, and tissue homogenatesXO activityPlate-based fluorometric measurement (ex 520-550 nm, em 585-595 nm)Based on a multistep enzymatic reaction in which the H2O2​ produced when XO oxidizes hypoxanthine reacts with ADHP
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DNA/RNA Damage

Item No.Product Name​Sample Type​MeasureMethodology​Additional Info
589320 DNA/RNA Oxidative Damage ELISA KitUrine, cell culture medium, cell lysates, tissue samples, saliva, and plasma/serum samples8-hydroxy-2’-deoxyguanosine, 8-hydroxy guanosine, and 8-hydroxy guaninePlate-based colorimetric measurement (405-420 nm)Monoclonal antibody (clone 15A3) used enables detection of 8-hydroxy-2’-deoxyguanosine (DNA oxidative damage marker), 8-hydroxy guanosine (RNA damage marker), and 8-hydroxyguanine (DNA/RNA damage marker) with selectivity and sensitivity highest for 8-hydroxy-2’-deoxyguanosine; does not correlate with LC/MS measurements of 8-hydroxy-2'-deoxyguanosine because the ELISA also detects 8-hydroxy guanosine and 8-hydroxy guanine
501130 DNA/RNA Oxidative Damage (Clone 7E6.9) ELISA KitUrine (other sample types have not been validated but may be used)8-hydroxy-2’-deoxyguanosine and 8-hydroxy guanosinePlate-based colorimetric measurement (405-420 nm)Monoclonal antibody (clone 7E6.9) used enables detection of 8-hydroxy-2’-deoxyguanosine (DNA oxidative damage marker) and 8-hydroxyguanosine (RNA damage marker) with equal selectivity and sensitivity; correlates with LC/MS measurements of a combination of 8-hydroxy-2'-deoxyguanosine and 8-hydroxy guanine
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Protein Oxidation (and Nitration)

Item No.Product Name​Sample Type​MeasureMethodology​Additional Info
600160 Methionine Sulfoxide Immunoblotting KitPlasma, CSF, cell/tissue lysates, or semi-pure/purified proteinsProteins containing MetO residuesImmunochemical detection by Western blotUtilizes a MetO polyclonal antibody isolated from rabbit serum that is specific for MetO and demonstrates minimal cross-reactivity with methionine sulfone
10010721 S-Glutathionylated Protein Detection KitWhole (permeabilized) cellsProtein-PSSG adductsStreptavidin-based detection by flow cytometry, fluorescence microscopy, or IP/avidin overlay analysisUtilizes a modified 'Biotin-switch' method to directly tag protein-PSSG adducts
10006518 S-Nitrosylated Protein Detection Kit (Biotin Switch)Whole cells or tissuesS-NO proteinsStreptavidin-based detection by Western blot or fluorescence microscopyUtilizes a modified 'Biotin-switch' method to directly tag S-NO proteins
601220 Nitrotyrosine IP KitCell lysatesNitrated tyrosine contentAffinity sorbent capture and elution by Western blot or proteomic analysisUtilizes a sorbent coupled with a nitrotyrosine monoclonal antibody to capture and pulldown nitrated proteins
10005020 Protein Carbonyl Colorimetric Assay KitPlasma, serum, urine, tissue homogenates, and cell lysatesProtein carbonyl contentPlate-based colorimetric measurement (360-385 nm)Utilizes the reaction between DNPH and protein carbonyls as a readout of protein oxidation
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Lipid Peroxidation

Item No.Product Name​Sample Type​MeasureMethodology​Additional Info
501140 DHN-MA EIA KitUrineDHN-MA, a 4-HNE metabolitePlate-based colorimetric measurement (405-414 nm)Compatible with human, mouse, rat, dog, and pig samples
516351 8-Isoprostane ELISA KitPlasma, serum, urine, lavage fluids, tissue homogenates, and culture medium8-IsoprostanePlate-based colorimetric measurement (405-420 nm)Overnight assay (incuabtion time = 18 hours) uses AChE tracer; assay range 0.8-500 pg/ml; detection limit (80% B/B0) of ~ 3 pg/ml
516360 8-Isoprostane Express ELISA KitPlasma, serum, urine, lavage fluids, tissue homogenates, and culture medium8-IsoprostanePlate-based colorimetric measurement (405-420 nm)4 hour assay uses AChE tracer; assay range 2.5-1,500 pg/ml; detection limit (80% B/B0) of ~ 10 pg/ml
500431 STAT-8-Isoprostane ELISA KitPlasma, serum, urine, lavage fluids, tissue homogenates, and culture medium8-IsoprostanePlate-based colorimetric measurement (405-420 nm)Extremely rapid assay (results in ~2 hours) uses an AP tracer; assay range 23.4-3,000 pg/ml; detection limit (80% B/B0) of ~ 45 pg/ml
705002 Lipid Hydroperoxide (LPO) Assay KitTissues, cultured cells, plant materials, foods, and biological fluidsLPOsPlate-based colorimetric measurement (500 nm)Designed for use with a single-tube spectrophotometer to read the results
705003 Lipid Hydroperoxide (LPO) Assay Kit (96 well)Tissues, cultured cells, plant materials, foods, and biological fluidsLPOsPlate-based colorimetric measurement (500 nm)Designed for use with a reusable glass plate
10009055 TBARS Assay KitPlasma, serum, urine, tissue homogenates, and cell lysatesMDA-TBA adductPlate-based colorimetric measurement (530-540 nm) or fluorometric measurement (ex 530 nm, em 550 nm)Standard method to determine lipid peroxidation; reaction yields higher sensitivity when measured fluorometrically, but a colorimetric method is included as an option
700870 TBARS (TCA Method) Assay KitPlasma, serum, urine, tissue homogenates, and cell lysatesMDA-TBA adductPlate-based colorimetric measurement (530-540 nm) or fluorometric measurement (ex 530 nm, em 550 nm)Offers the advantage of improved sample processing and reduced working volumes by incorporating a TCA precipitation procedure; maintains same reliability and accuracy as the original TBARS Assay; includes sample acid precipitation protocol to avoid confounding soluble TBARS
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Antioxidant Detection

Item No.Product Name​Sample Type​MeasureMethodology​Additional Info
709001 Antioxidant Assay KitPlasma, serum, urine, saliva, or cell lysatesTotal antioxidant capacityPlate-based colorimetric measurement (750 or 405 nm)Designed to measure the cumulative effects of all antioxidants present in plasma and body fluids, which offers more complete biological information compared to that obtained by the assessment of individual antioxidants
700420 Ascorbate Assay KitPlasma, serum, urine, and fruit juicesAscorbate (L-ascorbic acid/vitamin C)Plate-based fluorometric measurement (ex 340-350 nm, em 420-430 nm)Utilizes the condensation reaction of dehydroascorbic acid, the oxidation product of ascorbate, to determine ascorbate concentration
707002 Catalase Assay KitPlasma, serum, erythrocyte lysates, tissue homogenates, and cell lysatesCatalase activityPlate-based colorimetric measurement (540 nm)Utilizes the peroxidatic function of catalase to determine enzyme activity
700910 Catalase Assay Kit (without Hydrogen Peroxide)Plasma, serum, erythrocyte lysates, tissue homogenates, and cell lysatesCatalase activityPlate-based colorimetric measurement (540 nm)The 30% H2O2 solution needed to perform this assay is not included in this kit version as a shipping consideration to countries/institutions that regulate the receipt of such substances
600360 Glutathione Cell-Based Detection Kit (Blue Fluorescence)Cell lysatesIntracellular GSH levelsPlate-based fluorometric measurement (ex 380 nm, em 480 nm)Utilizes a cell-permeable dye that reacts with GSH to generate a fluorescent product that indicates intracellular level of GSH
703202 Glutathione Reductase Assay KitPlasma, cell lysates, and tissue homogenatesGR activityPlate-based colorimetric measurement (340 nm)Measures the rate of NADPH oxidation as a readout of GR activity
703302 Glutathione S-Transferase Assay KitPlasma, cell lysates, and tissue homogenatesTotal GST activity (cytosolic and microsomal)Plate-based colorimetric measurement (340 nm)Measures the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione as a direct readout of total GST activity
706002 Superoxide Dismutase Assay KitPlasma, serum, tissue homogenates, and cell lysatesCu/Zn, Mn, and Fe SOD activityPlate-based colorimetric measurement (440-460 nm)Measures the dismutation of superoxide radicals generated by XO and hypoxanthine as a readout of the activity of all three types of SOD
700340 Thiol Detection Assay KitPlasma, serum, urine, cell lysates, and tissue homogenatesFree thiol contentPlate-based fluorometric measurement (ex 380-390 nm, em 510-520 nm)Fluorometric detector reacts with free cysteine, glutathione, and cysteine residues on proteins to emit a fluorescent signal as a readout of thiol content


References

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9. Weimann, A., Broedbaek, K., Henriksen, T., et al. Free Radic. Res.46(4), 531-540 (2012).

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11. Oien, D.B., Canello, T., Gabizon, R., et al. Arch. Biochem. Biophys.485, 35-40 (2009).

12. Beckman, J.S. and Koppenol, W.H. Am. J. Physiol.271, C1424-C1437 (1996).

13. Jaffrey, S.R. and Snyder, S.H. Sci STKE2001(86), pl1 (2001).

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